Vectors

its applications & Limitations

Dr. Ujjal Kumar Nath
Professor
Department of Genetics & Plant Breeding
Bangladesh Agricultural University
Mymensingh
Vectors

A vector is a substance, usually a piece of DNA that carries a sequence of DNA or

other genetic material and introduces it into a new cell.

Functions:
v Vectors act as vehicles to transfer genetic material from one cell to the other for
multiplying, expressing, or isolation.
v Vectors are used as a tool in molecular cloning procedures to introduce the desired
DNA into a host cell.
v The DNA insert that is transmitted by a vector is termed recombinant DNA, and
the process is also known as recombinant DNA technology.
v Usually, the vectors are DNA sequences that carry different parts involved in
different functions. Vectors usually have an insert, which known as a transgene
that carries the recombinant DNA and a larger sequence called the backbone of the
vector responsible for the structure of the vector.
Functions:

v Vectors have particular features that carry the gene sequences

and enable them to survive within the host cell.

v Vectors are usually DNA sequences, viruses and other particles

can also function as vectors in processes like transduction.

v Vectors can be reused for multiple processes as these can be

recovered at the end of the process.

3
Characteristics or Features of vectors
1. Vectors should be capable of replicating autonomously, vector enables
to initiate replication and propagation within the host cell.
2. The size of an ideal vector should also be small enough to be
incorporated into the host genome.
3. Vectors should be easy to isolate and purify as these need to be
recovered and reused for multiple processes.
4. Most vectors have a gene that either provides resistance to an
antibiotic, which called marker genes.
5. Many vectors also require unique restriction enzyme recognition sites
that enable the insertion of the vector DNA.
6. The introduction of vectors into the host cell should be easy.
7. Vector is capable of integrating itself or the recombinant DNA into the
genome of the host cell.
8. Introduction of recombinant DNA into the vector doesn’t affect the
replication cycle of the vector.
4
Types of vectors
Vectors can be classified into different groups depending on the purpose of
the process and the type of particles used in the process. The following are
the commonly studied group of vectors;

1. Cloning vectors

2. Viral vectors

3. Expression vector

5
1. Cloning vectors
 Cloning vectors are vectors that are capable of replicating autonomously and used for the
replication of the recombinant DNA within the host cell.
 Cloning vectors are responsible for the determination of which host cells are appropriate for
replicating a particular DNA segment.
 Cloning vectors are of further different types that are defined by different features unique to
each type of vector.

6
a. Plasmid vector
q Plasmids are small extrachromosomal circular DNA molecules capable of
replicating autonomously within the host cell.
q Plasmids that makes them one of the best vectors is their small size. The small
size of the plasmid facilitates the separation of recombinant DNA from the host’s
genomic DNA.
q Plasmids can carry insert DNA that is less than 20 kb as the cloning efficiency
and plasmid stability decrease with the size of the vectors.
q The autonomous replication of plasmid is made possible by the presence of
genes and sequences that can initiate plasmid replication independent of the
host’s replication cycle.
q Bacterial plasmids contain ori sequences that not only control plasmid
replication but also determine the possibility of two plasmids coexisting within
the same host cell.
q Different plasmids have different types of selective markers, include antibiotic
resistance and the production of the β-galactosidase enzyme.
q Some of the most widely used plasmids are pBR322, pUC, and pBluescript
vectors that use E. coli as the host. 7
b. Cosmid
 Cosmid vectors are hybrid vectors composed of plasmid and phage λ vectors,
capable of incorporating up to 42 kb of DNA.

 Cosmid vectors are prepared by the insertion of the cos region of the phage vector
into the plasmid vectors.

 Cosmid vectors are large-sized vectors with sizes ranging from 400 base pairs to
30 kb. These can carry DNA sequences having sizes ranging from 28 to 46 kb.

8
 c. Bacteriophage vector

 Bacteriophage vectors are viruses that only infect bacteria and transform them
efficiently while carrying large inserts.
 Bacteriophages or phages have higher transformation efficiencies which increase
the chances of recovering a clone containing the recombinant DNA segments.
 The most important feature of a phage is the packaging system which enables the
incorporation of large eukaryotic genes and their regulatory elements.
 The use of phages also facilitates the isolation of larger quantities of DNA that
can be used for the analysis of the insert.

9
d. Bacterial artificial chromosome
 Bacterial artificial chromosomes are engineered DNA molecules that are used to clone
DNA segments in bacteria cells (usually E. coli).
 These consist of a bacteria-derived F-factor replication origin which enables the
propagation of large DNA fragments in a supercoiled circular form.

Bacterial artificial chromosomes can carry a much larger size of insert DNA as compared to
plasmid or phage vectors.

e. Yeast artificial chromosome

 Yeast artificial chromosomes are engineered DNA molecules that are used to clone DNA
inserts within the yeast cells, particularly Saccharomyces cerevisiae.
 YACs have been developed in order to clone large sequences of DNA so as to increase
the efficiency of the process.
 YACs can clone up to 500 kb of DNA, which is much higher than most traditional
cloning vectors.
 Even though these are frequently used as cloning vectors, they are also helpful in other
genetic processes like DNA sequencing and analysis.
10
f. Human artificial chromosome

 Human artificial chromosomes are extrachromosomal DNA fragments that act

as a new chromosome within the human cell.

 The use of human artificial chromosomes has increased with advances in

genetic engineering as it helps overcome problems commonly associated with
traditional vector systems.

 HACs can exist as single copy episomes without integration into the host
chromosomes allowing long-term stable maintenance.

11
2. Viral vectors
 Viral vectors are one of the most effective means of gene transfer to modify host cells or
tissues and manipulate them to express different types of genes.
 The concept of using viruses as vectors arose from the fact that viruses are very effective
in transducing their own genetic information into the host cell.
 During viral transduction, the non-essential viral genes are replaced with foreign DNA
sequences of therapeutic interest in order to produce recombinant viral vectors.
 Currently, different groups of viruses have been studied doe their possible use as viral
vectors to deliver genes to provide transient or permanent transgene expression.
 The use of viral vectors also enables location specificity with unique injection technology
within a specific time period.

12
3. Expression vector
q Expression vectors are vectors that enable the expression of cloned genes in order
to determine the successful cloning process.
q The use of expression vectors facilitates the processing of introns in prokaryotes as
these are designed with restriction sites next to the regulatory region.
q The restriction sites on the vectors result in splicing of the cloned gene to permit
the expression of the gene under the regulatory system.
q The regulatory system in expression vectors consists of a promoter sequence, a
termination sequence, along a transcription termination sequence.
q The use of expression vectors is essential to determine the success of a cloning
procedure and the efficiency of selective markers on the vectors.
q Expression vectors can be plasmid-based or viral-based that are introduced into the
host cells in order to code for particular mRNAs.
q The expression vectors are often used for the production of proteins that can then
be visualized by different methods depending on the complexity of the host cell.
q Expression vectors are of varying degrees of complexity depending on whether
they are to be used in prokaryotic or eukaryotic cells.
13
 Examples of Vectors

 pBR322 is a commonly used plasmid cloning vector used in prokaryotes, primarily E. coli.
 The vector consists of an origin of replication from a ColE1-like plasmid, pMB1, an ApR
gene (Ampicillin resistance gene) from the transposon, Tn3, and a TcR gene from pSC101.
 pBR322 was designed to overcome the limitations with pBR312 and pBR313, both of
which have extraneous DNA sequences and restriction enzyme cleavage sites that affected
their function as vectors.
 The structure of pBR322 was designed to maximize the number of restriction enzyme
cleavage sites on the vector and to minimize its size.
14
 The vector contains twenty-one unique restriction enzyme cleavage sites, eleven of which
are present in the TcR and ApR genes.
 The structure also facilitates a unique EcoRI cleavage site within the plasmid in order to
increase the efficiency of the vector.
 The pBR322 family of vectors was initially created for general cloning purposes in E. coli
and other similar prokaryotes; however, over the years, derivatives of the vector have
been designed for cloning purposes that specific to a particular organism or a particular
function.
 Even though pBR322 has been used for decades as an effective multipurpose cloning
vector, it has some limitations.
 The vector might be lost in continuous culture in the absence of selective pressure, which
might be a problem in large-scale fermentation of recombinant bacteria.

15
 pUC19 is also an example of a plasmid cloning vector that is used for the transfer of
recombinant DNA fragments into a host cell.
 The name ‘pUC19’ is given o the vector where the ‘p’ indicates plasmid and ‘UC’
indicates the University of California’ where the vector was designed and constructed.
 The vector is a double-stranded DNA molecule with 2686 bp and a high copy number.
 pUC19 consists of a 54 base-pair cloning site polylinker that further contains 13 different
hexanucleotide-specific restriction endonucleases.
 The colony screening after cloning with pUC19 is due to the presence of a selective
marker that encodes for the N-terminal fragment of β-galactosidase.
16
Applications of Vectors

The application of vectors in molecular biology and genetic engineering has

increased with time due to the simplicity, cost-effectiveness, and rapidity of the
process. The following are some of the major applications of vectors in molecular
biology;

1. Cloning vectors are the most important group of vectors that are used for the transfer of
foreign DNA into host cells for different purposes.
2. One of the most important applications of vectors is to generate engineered organisms
for a particular function, like engineering E. coli bacteria for insulin production.
3. Vectors can be used to isolate a particular gene sequence within a genome and to
determine its nucleotide sequence through DNA sequencing.
4. It also helps determine control sequences and regulatory sequences in genomes for their
study and analysis.
5. Cloning vectors can be used for studying the structure, function, and production of
protein in different organisms.

17
Applications of Vectors

6. Phage therapy is a form of therapy that uses bacteriophage vectors to treat

different bacterial infections in humans and other animals.
7. Vectors can also be used to identify mutations in different regions of DNA
sequences as well as to diagnose gene defects related to certain diseases.
8. Recombinant DNA technology has been used in clinical microbiology in
different approaches like recombinant antigens, recombinant vaccines, and
diagnostic probes.
9. Recombinant antigens prepared by cloning techniques by using cloning vectors
have been used for the screening of diseases like HIV, HCV, and CMV.
10. Vectors are one of the components in molecular biology which enable numerous
studies related to cell structure, nucleic acid composition, and genetic
engineering techniques.

18
Limitations of Vectors

1. Vectors are not very stable due to changes in metabolic energy and changing pH
and temperature in different hosts. The stability of vectors depends largely on the
type of vector and host genotypes.

2. Overexpression of a particular type of genes in the host cell is a common problem

associated with the use of vectors.

3. The use of a single type of vector might not be sufficient for a particular purpose.
The use of multiple vectors is complex and results in difficulties during the
process.

4. Even though a large number of studies are done in the field of molecular biology
for the production of more efficient vectors, it is a time-consuming and expensive
process.
19
Thank you

THANK YOU

20